LOW BARRIER HYDROGEN-BOND IS ABSENT IN THE CATALYTIC TRIADS IN THE GROUND-STATE BUT IS PRESENT IN A TRANSITION-STATE COMPLEX IN THE PROLYL OLIGOPEPTIDASE FAMILY OF SERINE PROTEASES

High frequency proton NMR spectra for two members of the prolyl oligop eptidase class of serine proteases, prolyl oligopeptidase and oligopep tidase B, showed that resonances corresponding to the active center hi stidine (NH)-H-delta 1 and (NH)-H-epsilon 2 generally observed in this region, are absent in these enzymes, However, for both enzymes, as we ll as with the H652A and H652Q active center variants of oligopeptidas e B, there are two resonances observed in this region that could be as signed to two protonated histidines with a noncatalytic function, The results indicate that these two histidines participate in strong hydro gen bonds, The absence of resonances pertinent to the active center hi stidine resonances suggests the absence of a low barrier hydrogen bond between the Asp and His in these two enzymes in their ground states, Addition of the peptide boronic acid t-butoxycarbonyl-(D)Val-Leu-(L)bo roArg to oligopeptidase B resulted in potent, slow binding inhibition of the enzyme and the appearance of a new resonance at 15.8 ppm, whose chemical shift is appropriate for a tetrahedral boronate complex and a low barrier hydrogen bond. The results demonstrate important dissimi larities between the active centers of the prolyl oligopeptidase class of serine proteases and the pancreatic and subtilisin classes both in the ground state and in the transition-state analog complexes.